1. Hypercoagulability and Thrombosis
Maria Domenica Cappellini
Erika Poggiali
University of Milan and Policlinico Foundation IRCCS Milan, Italy

2. Clinical challenges in NTDT
Iron overload
Hypercoagulability
Iron overload
Hypercoagulability
The 2 most important clinical challenges in patients with NTDT are iron overolad and hypercoagulability.
We focus our attention on hypercoagulability

3. Hypercoagulability
Why are we so concerned?

4. Epidemiology of thromboembolism in thalassaemia patients
Reference TI
n (%) TM
Type of thrombosis VT PE AT
Stroke
Zurlo et al., 1989 –
4/159 (2.5)
N/A
Michaeli et al., 1992
4/100 (4) *
Aessopos J et al., 1997
3/5 (60)
Moratelli et al., 1998
12/74 (16.2)
14/421 (3.3)
Borgna Pignatti et al., 1998
5/52 (9.6)
27/683 (4.0)
Cappellini et al., 2000
24/83 (29)
Borgna Pignatti et al., 2004
8/720 (1.1)
Early studies on Epidemiology, wide range of reported figures but the fact that TI > TM started being noticed.
Data on the epidemiology of TEE in beta-thal pts are limited, but evidence suggests that the incidence in pts with TI may be as musch as 4-fold higher than in pts with regularly transfused TM.
TI = β-thalassaemia intermedia; TM = β-thalassaemia major;
VT = venous thrombosis; PE = pulmonary embolism; AT = arterial thrombosis; N/A = not available.
Taher A, et al. Blood Rev 2008;22:

5. These data are confirmed by the largest clinical study from 8,860 pts form the Mediterrean area and Iran
Taher A, et al. Thromb Haemost. 2006;96:

6. Thromboembolic events
Patients (N = 8,860)
6,670 with TM
2,190 with TI
146 (1.65%) thrombotic events
61 (0.9%) with TM
85 (3.9%) with TI
Type of event
Risk factors for developing thrombosis in TI were
age (> 20 years)
previous thrombotic event
family history
splenectomy
Thromboembolic events in a large cohort of β-thalassaemia patients
The largest clinical study to date analyzed data from pts (6.670 TM, 2190 TI). The authors demonstrated that TEE occurred 4.38 times more frequently in TI than TM with more venous events occurring in TI and more arterial events occurring in TM.
Moreover, patients with TI who developed a TEE were more splencetomized, non-transfused, and had a hemoglobin level below 9 g/dL, which may justify a higher thrombotic drive as abonomrla RBCs are expected to remian longer in the circulation.
The study describe age beyind 20 years, splenectomy, family history of TEE and previuos TEE as the main risk factors dor develping thrombosis in the study group.
Thromboembolic events (%)
DVT = deep vein thrombosis; PE = pulmonary embolism; PVT = portal vein thrombosis; STP = superficial thrombophlebitis.
Taher A, et al. Thromb Haemost. 2006;96: 6 6

7. Can we redefine thalassaemia as a hypercoagulable state?

8. Pathophysiology of Thalassaemia
Extravascular haemolysis + Ineffective erythropoiesis
Release into the peripheral circulation of
damaged RBCs and erythroid precursors
The pathophisiology of TI is characterized by extravascular hemolysis and a high degree of ineffective erythropoiesis with the release into the peripheral circulation of damaged RBCs and erythroid precursors.
This has been attributed to severe complications such as pulmonary hypertension and thromboembolic phenomena.
Pulmonary hypertension (PHT)
and thromboembolic events (TEE)

9. Pathophysiology of thrombosis in NTDT
Cellular factors
Platelet activation
Pathology and alteration in red blood cells
Endothelial cells and peripheral blood activation (microparticles)
Nitric oxide
Splenectomy
Inherited and acquired coagulation defects
Other factors
Hyperoagulability in pts with NTDT has been attributed to several factors. It is often a combination of these factros that leads to clinical TEE.
Cappellini MD, et al. Ann N Y Acad Sci 2010;1202:231-6.

10. Hypercoagulability Hypercoagulability Nitric oxide
Other factors
Cardiac dysfunction
Hepatic dysfunction
Endocrine dysfunction
Nitric oxide
Hallmark of haemolysis
↓ Levels leading to vasoconstriction
Peripheral
blood elements
Expression of endothelial adhesion molecules and tissue factor on endothelial cells (ELAM-1, ICAM-1, vWF, VCAM-1)
Formation of microparticles
RBCs
Formation of reactive oxygen species
Expression of negatively charged phospholipids
Enhanced cohesiveness and aggregability
Hypercoagulability
Platelets
Increased platelet aggregation
Increased expression of activation markers
Presence of platelet morphologic abnormalities
Thrombophilia
No role for prothrombotic mutations
Decreased levels of antithrombin III, protein C, and protein S
Anti-phospholipid antibodies
Splenectomy
High platelet counts and hyperactivity
High levels of negatively charged RBCs
Cappellini MD, et al. Ann N Y Acad Sci 2010;1202:231-6.

11. Platelet activation Active thrombosis Chronic hypercoagulable state
Chronic platelet activation
↑ expression of CD62P
(P-selectin)
↑ expression of CD63
↓ platelet survival due to enhanced consumption
↑ platelet aggregation
Pts with beta-thal have activated PLTs. Flow cytometric studies confirmed the chronic PLT activation status.
In beta-thal., there is evidence of increased PLT aggregation, an increased proportion of PLT expressing CD62P (P-selectin) and CD63, and a shortened platelet survival due to ehnanced PLT consumption (especially in splenectomized pts).
Winichagoon P, et al. Asian J Trop Med Public Health 1981;12:556–60.
Del Principe D, et al. Br J Hematol 1993;84:111–7.
Ruf A, et al. Br J Hematol 1997;98:51–6.
Eldor A, et al. Am J Hematol 1989;32:94–9.

12. Pathology and alteration in red blood cells
Globin oxidation in erythroid cells (fastest in α-globin chains)
Hemichromes
Bind to membrane band 3, ankyrin and spectrin
Heme disintegrates
Toxic nontransferrin bound iron
Reactive oxygen species
Iron-dependant oxidation of membrane proteins
Senescence antigens (phosphatidyl-serine)
Rigid, deformed RBCs aggregate and are prematurely removed
Splenectomy favours persistence of these RBCs in the circulation
Thalassemic RBCs with negatively charged phopholipids increase thrombin generation. A high number of circulating RBCs with negatively charged phopholipids was found in splenectomized pts.

13. peripheral blood activation
Endothelial cells and
peripheral blood activation
↑ELAM-1
↑ICAM-1
↑VWF
↑E-selectin
↑VCAM-1
Vessel Obstruction
Tissue Hypoxia
Tissue Death
Haemolysis
The role of peripheral blood activation is documented by the increased expression of … …. … that suggest that endothelial injury or activation may be a feature of the disease and can aid the recruitment of WBCs and PLTs promoting thrombosis.
Butthep P, et al. Thromb Hemost 1995;74:1045–9.
Butthep P, et al. Southeast Asian J Trop Med Public Health 1997;28(Suppl. 3):141A–8A.
Hovav T, et al. Br J Hematol 1999;106:178–81.

14. Hypercoagulability Hypercoagulability Nitric oxide
Other factors
Cardiac dysfunction
Hepatic dysfunction
Endocrine dysfunction
Nitric oxide
Hallmark of haemolysis
↓ levels leading to vasoconstriction
Peripheral
blood elements
Expression of endothelial adhesion molecules and tissue factor on endothelial cells (ELAM-1, ICAM-1, vWF, VCAM-1)
Formation of microparticles
RBCs
Formation of reactive oxygen species
Expression of negatively charged phospholipids
Enhanced cohesiveness and aggregability
Hypercoagulability
Thrombophilia
No role for prothrombotic mutations
Decreased levels of antithrombin III, protein C, and protein S
Anti-phospholipid antibodies
Platelets
Increased platelet aggregation
Increased expression of activation markers
Presence of platelet morphologic abnormalities
Splenectomy
High platelet counts and hyperactivity
High levels of negatively charged RBCs
Splenectomy: can contritbute to increase susceptibility to thrombosis and it is linked to high PLT counts and aggregation following splenectomy, and(or to increased number of damaged RBCs.
Thrombophilia: does not have a role, even if decreased levels of ATIII, protein C and S, and high levels of anti-phosholpid antibodies have been documented, the exact nature of these antibodies and their relation to coexistent hepatitis C virus is still under investigation.
Cappellini MD, et al. Ann N Y Acad Sci 2010;1202:231-6.

15. The epidemiological data and the clinical experience

16. OPTIMAL CARE study: incidence and risk factors for thrombosis
Parameter
Frequency
n (%)
Age (years)
< 18
172 (29.5 )
18–35
288 (49.3)
> 35
124 (21.2)
Male : Female
291 (49.8) : 293 (50.2)
Splenectomized
325 (55.7)
Serum ferritin (µg/L)
< 1,000
376 (64.4)
1,000–2,500
179 (30.6)
> 2,500
29 (5)
Complications
Osteoporosis
EMH
Hypogonadism
Cholelithiasis
Thrombosis
Pulmonary hypertension
Abnormal liver function
Leg ulcers
Hypothyroidisim
Heart failure Diabetes mellitus
134 (22.9)
101 (17.3)
100 (17.1)
82 (14)
64 (11)
57 (9.8)
46 (7.9)
33 (5.7)
25 (4.3)
10 (1.7)
Complication
Parameter
RR*
95% CI
p value
Thrombosis
Age > 35 years
2.60
0.003
Hb ≥ 9 g/dL
0.41
0.001
Serum ferritin ≥ 1,000 µg/L
1.86
0.023
Splenectomy
6.59
< 0.001
Transfusion
0.28
*RR indicates adjusted relative risk.
High rate of thrombosis confirmed
EMH = extramedullary haematopoiesis; CI = confidence interval.
Taher AT, et al. Blood. 2010;115:

17. Frequency of thrombosis increases with age in NTDT patients
N = 120 treatment-naive* TI patients
< 10 years
11–20 years
21–32 years
> 32 years *
* = statistically significant trend * * * * *
*never received any treatment intervention (splenectomy, transfusion,
iron chelation therapy, or HbF-inducing agents). PHT = pulmonary hypertension; HF = heart failure; ALF = abnormal liver function; DM = diabetes mellitus.
Taher A, et al. Br J Haematol 2010;150:486-9.

18. High prevalence of thromboembolic events, particularly in splenectomized patients
Thrombin-generated (nM) 10 30 60 90
120
150
Splenectomized patient with TI
Non-splenectomized patient with TI
Non-splenectomized controls
Splenectomized controls
Time (s)
Thromboembolic events occurred in 24/83 (29%) transfusion-independent patients with TI who had undergone splenectomy
Cappellini MD, et al. Br J Haematol. 2000;111:

19. OPTIMAL CARE study: patient stratification according to splenectomy and TEE status
Three groups of patients identified
Group I, splenectomized patients with a documented TEE (n = 73)
Group II, age- and sex-matched splenectomized patients without TEE (n = 73)
Group III, age- and sex-matched non-splenectomized patients without TEE (n = 73)
Type of thromboembolic event in splenectomized TI patients (Group I)
n (%)
DVT
46 (63.0)
PE*
13 (17.8)
STP
12 (16.4)
PVT
11 (15.1)
Stroke
4 (5.5)
*All patients who had PE had confirmed DVT.
TEE = thromboembolic events; DVT = deep vein thrombosis; PE = pulmonary embolism; STP = superficial thrombophlebitis; PVT = portal vein thrombosis.
Taher A, et al. J Thromb Haemost. 2010;8:

20. OPTIMAL CARE study: patient stratification according to splenectomy and TEE status (cont.)
Parameter
Group I
Splenectomized with TEE
(n = 73)
Group II
Splenectomized without TEE
Group III
Non-splenectomized
p value
Mean age ± SD, years
33.1 ± 11.7
33.3 ± 11.9
33.4 ± 13.1
0.991
Male : female
33 : 40
35 : 38
34 : 39
0.946
Mean Hb ± SD, g/dL
9.0 ± 1.3
8.8 ± 1.2
8.7 ± 1.3
0.174
Mean HbF ± SD, %
45.9 ± 28.0
54.4 ± 32.8
44.2 ± 27.2
0.429
Mean NRBC count ± SD, x106/L
436.5 ± 205.5
279.0 ± 105.2
239.5 ± 128.7
< 0.001
Mean platelet count ± SD, x109/L
712.6 ± 192.5
506.3 ± 142.1
319.2 ± 122.0
PHT, n (%)
25 (34.2)
17 (23.3)
3 (4.1)
HF, n (%)
7 (9.6)
5 (6.8)
1 (1.4)
0.101
DM, n (%)
4 (5.5)
0.256
Abnormal liver function, n (%)
2 (2.7)
0.863
Family history of TEE
3 (4.7)
0.554
Thrombophilia, n (%)
Malignancy, n (%)
0 (0)
0.363
Transfused, n (%)
32 (43.8)
48 (65.8)
54 (74.0)
0.001
Anti-platelet or anticoagulant use, n (%)
0.598
Hydroxyurea use, n (%)
13 (17.8)
29 (27.4)
0.383
Splenectomized patients with a documented TEE had significantly higher NRBC (≥ 300 x 106/L), platelets (≥ 500 x 109/L), pulmonary hypertension occurrence, and were mostly non-transfused
Hb = total haemoglobin; HbF = fetal haemoglobin; NRBC = nucleated red blood cell; PHT = pulmonary hypertension; HF = heart failure; DM = diabetes mellitus.
Taher A, et al. J Thromb Haemost. 2010;8:

21. Time-to-thrombosis (TTT) since splenectomy
The median TTT following splenectomy was 8 years (range 1–33 years)
Taher A, et al. J Thromb Haemost. 2010;8:

22. Time-to-thrombosis (TTT) since splenectomy (cont.)
0.2
0.4
0.6
0.8 1 5 10 15 20 25 30 35 40
NRBC count
< 300 x 106/L
≥ 300 x 106/L
0.2
0.4
0.6
0.8 1 5 10 15 20 25 30 35 40
Platelet count
< 500 x 109/L
≥ 500 x 109/L
Cumulative thrombosis-free survival
Cumulative thrombosis-free survival
Duration since splenectomy (years)
Duration since splenectomy (years)
0.2
0.4
0.6
0.8 1 5 10 15 20 25 30 35 40
There was no statistically significant difference in the median TTT between patients with and without PHT (9 vs. 8 years, P = 0.703)
Transfused
Yes No
0.2
0.4
0.6
0.8 1 5 10 15 20 25 30 35 40
Pulmonary hypertension
Yes No
Cumulative thrombosis-free survival
thrombosis-free survival
Cumulative
Duration since splenectomy (years)
Duration since splenectomy (years)
Taher A, et al. J Thromb Haemost. 2010;8:

23. Clinical recommendations for the prevention of thromboembolic events
A guarded approach to splenectomy in β-thalassaemia patients is recommended unless strongly indicated
In already-splenectomized NTDT patients, those at high risk of thrombosis may be identified early by their high NRBC and platelet counts, evidence of PHT, and transfusion naivety
attention should also be paid to the aging NTDT patients
Prospective clinical trials that evaluate the efficacy, safety, and cost-effectiveness of transfusions and anti-platelet and anticoagulant therapy in preventing thromboembolism are necessary
aspirin for stroke prevention and lifelong anticoagulation treatment in patients with a history of thrombotic events
Taher AT, et al. Br J Haematol. 2011;152:

24. OPTIMAL CARE study: transfusion therapy reduces the risk of complications
Parameter RR
95% CI
p value
EMH
Splenectomy
0.44
0.26–0.73
0.001
Transfusion
0.06
0.03–0.09
< 0.001
Hydroxyurea
0.52
0.30–0.91
0.022
Pulmonary hypertension
Age > 35 years
2.59
1.08–6.19
0.032
4.11
1.99–8.47
0.33
0.18–0.58
0.42
0.20–0.90
0.025
Iron chelation
0.53
0.29–0.95
Heart failure
0.02–0.17
Thrombosis
2.60
1.39–4.87
0.003
Hb ≥ 9 g/dL
0.41
0.23–0.71
Serum ferritin ≥ 1,000 µg/L
1.86
1.09–3.16
0.023
6.59
3.09–14.05
0.28
0.16–0.48
Cholelithiasis
2.76
1.56–4.87
Female
1.96
1.18–3.25
0.010
5.19
2.72–9.90
0.36
0.21–0.62
0.30
0.18–0.51
Abnormal liver function
1.74
1.00–3.02
0.049
n = 445 occasionally/regularly transfused patients (N = 584)
Taher AT, et al. Blood. 2010;115:

25. OPTIMAL CARE study: transfusion therapy reduces the risk of complications (cont.)
Parameter RR
95% CI
p value
Leg ulcers
Age > 35 years
2.09
1.05–4.16
0.036
Splenectomy
3.98
1.68–9.39
0.002
Transfusion
0.39
0.20–0.76
0.006
Hydroxyurea
0.10
0.02–0.43
Hypothyroidism
6.04
2.03–17.92
0.001
0.05
0.01–0.45
0.003
Osteoporosis
3.51
2.06–5.99
< 0.001
Female
1.97
1.19–3.27
0.009
4.73
2.72–8.24
3.10
1.64–5.85
0.02
0.01–0.09
Iron chelation
0.40
0.24–0.68
Hypogonadism
2.98
1.79–4.96
Serum ferritin ≥ 1,000 µg/L
2.63
1.59–4.36
16.13
4.85–52.63
4.32
2.49–7.49
2.51
1.48–4.26
Transfusion therapy was protective for thrombosis, EMH, PHT, HF, cholelithiasis and leg ulcers
Transfusion therapy was associated with an increased risk of iron overload-related endocrinopathy
n = 445 occasionally/regularly transfused patients (N = 584).
Only significant associations presented.
Taher AT, et al. Blood. 2010;115:

26. Indications of RBC transfusion in TI
Hb < 5 g/dL
Declining Hb level with progressive splenic enlargement (> 3 cm/year)*
Poor growth and/or development
Evidence of
bone deformities
clinically relevant tendency to thrombosis
leg ulcers
EMH
PHT
infections
Prior to surgical procedures
Pregnancy
Many patients require intermittent RBC transfusions due to intermitent conditions (infection, surgery), and more regular therapy is often indicated for growth failure, bone deformities or when Hb levels decline due to progressive splenomegaly.
Hb = heamoglobin.
* At least in periods of maximal growth and development.
Taher A. et al. Blood Reviews 26S (2012); S24-S27. 26

27. Initiating transfusion therapy in NTDT patients
The decision to initiate transfusion in these patients is very difficult due to the heterogeneity of the disease
There is no benefit in limiting the quantity or frequency of transfusions once they have begun
Starting transfusions after the third year of life has been shown to increase the risk of alloimmunization
Transfused patients with TI experience fewer TEE, PHT and silent brain infarcts compared to transfusion-naïve patients, due to the correction of the ineffective erythropoiesis and resulting damaged RBCs with thrombogenic potential
An individualized approach is recommended to estabilish an optimal strategy for preventing the occurrence of TEEs.
There may be clinical benefit to initiating transfusions earlier or phrophylactically to reduce the risk of allommunization and to prevent complcations that can occurr with delayed initiation of transfusions.
Taher A. et al. Blood Reviews 26S (2012); S24-S27.

28. Summary Thromboembolic events are frequent in β-thalassaemia patients
oxidative damage to RBCs, impacting their membrane properties, resulting in increased aggregation and risk of thromboembolism
risk of thromboembolism increases with age, and is influenced strongly by splenectomy and transfusion navïety
Splenectomy is associated with a high risk of thrombosis, particularly in patients with high NRBC or platelet counts, who are transfusion-naïve
Transfusion therapy reduces the risk of thrombosis in NTDT patients
transfusion iron intake inevitably increases the risk of iron overload, but the
benefit of transfusion therapy may greatly outweigh the cost and inconvenience
of iron chelation therapy
Many different factors can contribute to the hypercoagulability state observed in pts with beta-thalassemia.
In most cases, a combination of these abnormalities leads to clinical thrombosis.
The higher incidence of thrombotic events in TI compared to TM is mainly attributed to transfusion naivety and splenectomy, both of which promote an underlying procoagulant activity.

29. Summary
Despite various treatment options are available, no clear guidelines exist: each patient must be assessed individually and assigned a personalized thrombotic risk based on intrinsic and extrinsic factors
Several studies are highlighting the roles of transfusion, iron chelation therapy, and fetal haemoglobin induction (hydroxycarbamide, HU) in the management of NTDT; thus these approaches merit large prospective evaluation
Another approach would be to correct the reactive oxygen species-induced RBC membrane damage using antioxidants, although this approach has not yet been verified in clinical trials
No clear guidelines exist to estabilish a prophylactic strategy: an individualized approach that takes into consideration all associated risk factors is advisable.
It may also possible to design thalassemia-tailored thrombosis risk-assessment model (RAM) to estimate thrombotic risk as a function of intrinsci (e.g. thalassemia type and number of circulating RBC) and extrinsic (surgery, infection, splenectomy) factors.
Hydoroxicarbamide may decrease coagulation activation by reducing phopholipid expression on the surface of both RBCs and PLTs, and decreasing RBC adhesion to thrombospondin.

30. Acknowledgments
I would like to thank you for the attention, and all the Congenital Anemia Center Staff for their support
Prof. M.D. Cappellini
Giovanna Graziadei Irene Motta
Alessia Marcon Ilaria Gandolfi
Laura Zanaboni Marianna Giuditta
Elena Cassinerio Marta Mazzoleni
Claudia Cesaretti Silvio De Fazio
All the nursing staff
Growth differentiation factor 15 (GDF15) and twisted gastrulation 1 (TWSG1) released by erythroblasts have been proposed to be involved in hepcidin suppression. In patients with homozygous β-thalassemia or other anemias with ineffective erythropoiesis, elevated serum GDF15 correlates with diminished hepcidin levels and increased iron absorption.

32. Alteration of the phospholipid “Flip-Flop” mechanism:
RBCs with negatively charged phospholipids
Adherence of RBCs to endothelial cells is increased
Phosphatidylserine on damaged or senescent RBCs leads to
Recognition by phagocytes
Removal from circulation
Apoptosis
Splenectomy favours persistence of these RBCs in the circulation
Thrombus formation
Fibrin/ platelets
Thrombin generation
Courtesy of Dr A. Taher.